Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450_scc) to form pregnenolone. Luteal mRNA encoding P450_scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450_scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450_scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F₂ (PGF₂). P450_scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF₂ (P < 0.05). An assay for P450_scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450_scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450_scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450_scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.